INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Most natural aquatic and terrestrial environments are nutrient limited and, as a result, a major portion of the biosphere exists as oligotrophic (nutrient-depleted) habitats (22, 32). Of all the environments on Earth, however, the ocean has the highest cellular production rate (36). The number of prokaryotes in the ocean is estimated to be 1.2 × 10²⁹ (36) and is mainly due to the growth of oligotrophic, extremely small bacteria (i.e., ultramicrobacteria), a unique class of marine bacteria that proliferate by growing slowly, even at nanomolar concentrations of growth substrates (reviewed in reference 32).

Although ultramicrobacteria represent the ocean's main bacterial component in terms of both activity and biomass (32) and although they also predominate in soil ecosystems (19), their physiology has remained largely uncharacterized (19). The lack of knowledge largely relates to the difficulty in isolating them from the environment. In 1990, an important breakthrough was made with the isolation of the ultramicrobacterium Sphingomonas sp. strain RB2256 (herein referred to as RB2256) from Resurrection Bay, Alaska (6, 29). At the time of isolation, it was a numerically dominant bacterium. Morphologically (29) and phylogenetically (M. Vancanneyt, F. Schut, C. Snauwaert, J. Goris, J. Swings, and J. C. Gottschal, submitted for publication) related bacteria have also been isolated as dominant species from the North Sea, and two years after initial isolation, RB2256 was again isolated as one of the dominant species in Resurrection Bay (M. Vancanneyt et al., submitted). Recently, a phylogenetically related strain was also isolated from waters near Japan (M. Eguchi et al., unpublished data).

Characteristics that define RB2256 as a typical or model oligotrophic ultramicrobacterium (15, 18, 32, 33) include a constant ultramicrosize (<0.1 µm³), irrespective of whether it is growing or starved; a mechanism for avoiding predation (ultramicro size), a relatively slow maximum specific growth rate (<0.2 h¹); the ability to utilize low concentrations of nutrients; high-affinity, broad-specificity uptake systems; and the ability to simultaneously take up mixed substrates (8, 10, 29-31). Based on the Michaelis-Menten constant for substrate transport (K_t) and the available concentrations of mixed amino acids in the ocean, RB2256 is predicted to have an in situ doubling time of 12 h to 3 days (6, 29). As the average doubling time for microorganisms in oligotrophic waters is estimated to be 5 to 15 days (14), RB2256 is likely to be a significant contributor to biomass turnover in oligotrophic ocean waters.

A fundamental characteristic that distinguishes RB2256 from typical copiotrophic bacteria is the capacity for copiotrophic bacteria to grow rapidly in the presence of high concentrations of nutrients and to undergo reductive cell division to form resting-stage cells when exposed to nutrient deprivation (22, 34). This may be referred to as a feast-and-famine response. As RB2256 is well suited for growth in low concentrations of nutrients, it may be expected that it would rarely encounter nutrient levels that would cause starvation, and as a result it may not have the genetic potential to respond to complete nutrient deprivation in the same way as copiotrophic bacteria.

The potential for RB2256 to starve may, however, be considered in terms of the total bacterial carbon content versus the amount of carbon available for microbial assimilation. Bacterial growth in the subarctic Pacific Ocean is limited by carbon (20), and approximately 1 to 10% of dissolved organic carbon (DOC) is available for microbial assimilation (22). This mainly consists of dissolved free amino acids and carbohydrates. The greatest proportion of bioavailable carbon in these oligotrophic waters consists of dissolved free amino acids at a concentration of 3 to 191 nM (9, 23) or 0.1 to 8.8 µg of C liter¹ (based on 110 as the average molecular weight for an amino acid and a ratio of 25/60 for conversion to carbon). Assuming an average bacterial cell carbon content of 5.6 × 10¹³ g of C µm³ (4) and an average cell volume of RB2256 of 0.05 µm³ (10), the carbon content is 28 fg of C cell¹. At the upper limit of bioavailable amino acids (8.8 µg of C liter¹), the cell yield is 3 × 10⁵ cells ml¹. As RB2256 was isolated from a 10⁶-fold dilution series of seawater from Resurrection Bay in which the standing bacterial population was approximately 2 × 10⁵ cells ml¹ (32), this would indicate that the cells were likely to be starving at the time of sampling. There are possible limitations associated with this analysis, including uncertainties regarding measurements of bioavailable DOC and diurnal and seasonal variations (23); however, as an approximation, it indicates the potential for bacteria in oligotrophic marine environments to be exposed to starvation.

In order to assess the capacity of RB2256 to respond to starvation, we compared gene expression in logarithmic and starved cells using a two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of cellular proteins. The generation of high-resolution protein profiles enabled a quantitative assessment of differential gene expression in RB2256 and a comparison of gene expression to that of Vibrio angustum S14 (26), a typical marine copiotrophic bacterium with a well-characterized response to starvation (34). In addition to patterns of whole-cell gene expression, we examined changes in total protein and RNA synthesis that occurred as the cells made the transition from logarithmic growth to starvation. By monitoring the response throughout the growth phase and up to 7 days of starvation, we have gained significant insight into how the cell prepares for and copes with starvation. Our findings demonstrate that RB2256 exhibits a defined response to starvation with a number of characteristics that differ from copiotrophic bacteria. These results expand our view of the physiology of oligotrophic bacteria, and we discuss the potential advantages this may have for bacterial survival under oligotrophic conditions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Organism, growth conditions, and media. Sphingomonas sp. strain RB2256 is a member of the -proteobacteria. It has recently been taxonomically and phylogenetically characterized, and the species name Sphingomonas alaskensis sp. nov. has been proposed (M. Vancanneyt et al., submitted). RB2256 was maintained in an artificial seawater medium (10) supplemented with 3 mM D-glucose (ASWG medium). Batch cultures were grown at 30°C with orbital shaking at 150 rpm, as described previously (11). Cell viability was monitored by the drop plate method (13) with VNSS solid medium (25).

Determination of glucose concentration in growth medium. RB2256 cultures were grown in ASWG medium, and samples were filtered through sterile 0.22-µm-pore-size Millex-GV (Millipore) membranes. Filtrates were analyzed with a glucose oxidase-peroxidase kit according to manufacturer's protocols (Sigma Chemical Co., St. Louis, Mo.).

Determination of rates of protein and RNA synthesis during growth and starvation. The rates of protein and RNA synthesis were determined as the rate of radioactive methionine-cysteine and uridine incorporation into tricholoroaceteic acid (TCA)-insoluble material, respectively (13). Cultures were monitored by measuring the optical density at 433 nm (OD₄₃₃) (11). Cultures were sampled during logarithmic growth, at the onset of starvation, and 1, 2, 4, and 7 days thereafter and exposed to a final concentration of 0.06 µM [³⁵S]methionine-cysteine (specific activity, 1,175 Ci mmol¹; ICN Pharmaceuticals) or 0.38 µM [³H]uridine (specific activity, 26 Ci mmol¹; Amersham Life Science) at 30°C for up to 60 min. Initial rates were calculated from the linear portion of graphs of incorporation versus time. To determine synthesis rates, 50-µl duplicate samples were removed and added to 800 µl of ice-cold 10% TCA and maintained on ice until all samples were collected. Samples used for determining rates of protein synthesis were heated at 90°C for 15 min, kept on ice for 1 h, and collected by filtration with 0.2-µm-pore-size filters (Millipore). Samples used for the determination of rates of RNA synthesis were filtered without prior heat treatment. Filters for both protein and RNA determinations were washed three times with ice-cold 5% TCA and transferred into scintillation vials, and 10 ml of scintillation cocktail (Wallac Hi-Safe 3) was added. Filters were mixed by vortexing for 40 s prior to measuring radioactivity (Packard Scintillation Counter Series 2000). Rates of protein and RNA synthesis were calculated from the linear slopes of radionuclide incorporation graphs (25). The rate (in disintegrations per minute per cell) was calculated from the slope (disintegrations per minute), the viable cell count (the number of cells per milliliter), and sample volume (in milliliters). The rate was converted to the number of picomoles per minute per cell by incorporating the specific activity (curies per millimole) for each radionuclide and conversion factor (1 Ci = 2.2 × 10¹² dpm) and converting to the number of picomoles per minute per milligram of protein by using the measured protein content per cell (see below). Relative rates were calculated as the proportion of the maximum rate during logarithmic growth and are represented as percentages or decimal values.

Determination of cellular protein content. Cultures were sampled (15-ml samples) during logarithmic growth, at the onset of starvation, and 1, 2, 4, and 7 days thereafter and collected by centrifugation at 2,570 × g at 25°C for 20 min (Hettich Universal 16R). The cell pellet was resuspended in 15 ml of 0.1% sodium dodecyl sulfate by brief vortex mixing, followed by dilution with an equal volume of sterile Milli-Q water. Protein concentrations were determined with a bicinchoninic acid assay kit in accordance with the manufacturer's protocols (Sigma Chemical Co.), with standard curves constructed with bovine serum albumin.

Determination of ribosome content. Data for ribosomes was determined previously (11).

Separation and analysis of 2D-PAGE of proteins. Proteins from cells in mid-logarithmic growth (OD₄₃₃ = 0.17; 1.5 × 10⁸ cells ml¹) or following 24 h of starvation were radioactively labelled with [³⁵S]methionine-cysteine (ICN Pharmaceuticals). Pulse-labelling, sample preparation, and electrophoresis were performed as described previously (12). Images of 2D-PAGE gels were obtained with CS phosphor screens on a Bio-Rad GS525 Molecular Imager. Quantitative analysis of 2D-PAGE gels was performed using Melanie II software (Bio-Rad) with three images generated from two separate experiments for each growth condition. Logarithmic-phase cells were labelled for 60 min (12). Starved cells were labelled for various lengths of time to determine the period of exposure that provided an equivalent level of incorporation per cell. By comparing disintegrations per minute from whole-cell counts with liquid scintillation and total disintegrations per minute from 2D-PAGE gels, equivalent levels of incorporation were found after 12-h labelling of cells starved for 24 h. Each spot was allocated a unique identification number. After initial spot detection, landmarks were manually appointed on each image, and all data were matched automatically with Melanie II software. Each spot that was identified by the spot-finding program was manually examined to confirm that it was a real spot. Anomalous spots, including regions of streaking or smearing and regions at the extremities of the gel, were excluded. Each gel was compared to every other gel to identify spots they had in common. Every match between spots from any two gels was designated a group. For each growth condition, groups were visually examined to ensure that the analogous spot was detected in all three gels. Anomalous spots were excluded or manually adjusted. Groups between different growth conditions were statistically analyzed (Student t test, 95% confidence interval) to determine which groups had intensity differences of at least twofold. Spots which appeared in the three gels from one growth condition (e.g., logarithmic growth) but did not appear in any of the three gels from another growth condition (e.g., starvation) were designated unique to the particular growth condition (e.g., logarithmic growth).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

In order to examine the physiological changes that take place in stationary phase, we determined rates of RNA and protein synthesis, protein content per cell, and the glucose concentration of the growth medium throughout the growth phase and starvation and generated two-dimensional protein profiles of logarithmic-phase cells and cells starved for 24 h.

Onset of starvation and glucose utilization. During growth at 30°C in ASWG medium, maximum growth yields corresponded to an OD₄₃₃ of 0.8 (Fig. 1A). Once this optical density was reached, it did not increase further. In contrast, viable cell numbers increased from 1.4 × 10⁹ CFU ml¹ when OD₄₃₃ first reached 0.8 to 2.2 × 10⁹ CFU ml¹ over the next 9 h. In association with cell growth, the glucose concentration decreased during log phase to a relative level of 0.023 when the cells reached an OD₄₃₃ of 0.8. Within the next 5 h, the glucose level continued to fall to a relative level at least as low as 0.003 (the detection limit of the glucose assay).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Growth phase-dependent, macromolecular synthesis in RB2256. Cells were grown in ASWG medium. (A) Cell growth is measured as OD433 (), as the number of CFU per milliliter (*), and as the concentration of glucose in the medium (). Curves for optical density and glucose concentration are the averages of two experiments with a maximum standard deviation of less than 20%, and results of a typical experiment are shown for the number of CFU per milliliter. (B) The optical density of cultures () and the relative rate of RNA synthesis () are shown. The results of a typical experiment are shown. (C) The optical density of cultures (), the relative number of ribosomes per cell (), protein content per cell (), and rates of protein synthesis () are shown. The vertical hatched bar highlights the logarithmic-phase time point used for 2D-PAGE pulse-labelling experiments (see Fig. 3). Curves for optical density and protein content per cell are results of a typical experiment, rates of protein synthesis are the average of two experiments with a standard deviation of less than 27%, and ribosome numbers are from Fegatella et al. (11). All experiments were performed a minimum of three times.

1

1

1

RNA synthesis during growth. Peak rates of RNA synthesis occurred during late logarithmic phase (Fig. 1B). Prior to the observed increase in the rate of RNA synthesis, rates of RNA synthesis were relatively constant (54 to 66% of maximal rates), and following the peak, synthesis rates decreased rapidly to a relative level of around 16% at the onset of starvation. This indicated that RNA synthesis rates remained at relatively high levels during the most active stages of growth and that synthesis was quickly down-regulated as the cells approached starvation. The highest rates of RNA synthesis were 1.0 × 10⁸ pmol of uridine min¹ cell¹.

Protein synthesis during growth. The rate at which proteins were synthesized increased rapidly during logarithmic phase, peaked around late logarithmic phase, and decreased rapidly to reach a minimal basal level as the cells approached the onset of starvation (Fig. 1C). The rate of protein synthesis increased from a relative level of approximately 2% and decreased from peak levels to 2% at the onset of starvation, demonstrating that protein synthesis was regulated (gene expression and protein turnover) by up to 50-fold throughout the growth phase. Highest rates of protein synthesis were 5.2 × 10⁸ pmol of methionine min¹ cell¹ and 284 pmol of methionine min¹ mg of protein¹ (Table 1). The profile describing the protein content per cell had a peak that coincided with the peak in the rate of protein synthesis. However, the profile of the rate of protein synthesis was broad, with relative levels around 1 day prior to starvation of 17% and 56% at the onset of starvation (Fig. 1C). The maximum concentration of protein corresponded to 186 fg cell¹ (Table 1).

1

1

Ribosome content and protein and RNA synthesis. The number of ribosomes per cell during logarithmic growth (11) is shown in comparison to rates of protein synthesis and protein content (Fig. 1C). The peak in ribosome content occurred much earlier in the growth phase than the peaks for the protein synthesis and protein content per cell. At the point in the growth phase when the maximum number of ribosomes were present (2,000 per cell [11]), the total protein content was 45 fg cell¹ (data not shown). Assuming that the ribosomes in RB2256 have a mass equivalent to that of Escherichia coli (857,000 [21]), the protein content of the ribosomes at this point was 2.9 fg cell¹ or approximately 6% of total cell protein. This compares to 9% for E. coli at a specific growth rate of 0.6 h¹ (5). By applying this same calculation to time points from the onset to 7 days of starvation (Fig. 2), ribosomal protein represented less than 0.9% of total protein.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Macromolecular synthesis in RB2256 during starvation. Conditions for the experiments are as described in the legend to Fig. 1. Cell growth was measured by determining OD433 (), and protein content per cell (), relative number of ribosomes per cell (), relative rate of RNA synthesis (), and rates of protein synthesis () are shown.

Macromolecular synthesis during starvation. During stationary phase, up to 7 days of starvation, rates of RNA (0.3 to 2%) and protein (0.3 to 0.9%) synthesis reached low levels that remained constant throughout the starvation period (Fig. 2). The protein and ribosome contents in the cell also remained constant throughout starvation; however, the relative levels were approximately 25 and 10%, respectively. The protein content has also been found to remain high (50.4 mg of protein liter¹) for glucose-limited chemostat cells that have been starved for up to 29 days (28). These data show that low basal levels of macromolecular synthesis occurred during starvation and that preformed ribosomes and whole cell protein were retained for at least 7 days.

Protein profiles. High-resolution 2D-PAGE profiles were generated for cells in mid-logarithmic growth (1.5 × 10⁸ CFU ml¹) and after 24 h of starvation. The time point for logarithmic growth corresponded to 0.4 day before the onset of starvation and prior to the major increases in rates of RNA and protein synthesis (Fig. 1). This time was chosen as it was expected to reveal greater differences in expression between logarithmic growth and starvation than if a later time point was chosen for logarithmic growth. These profiles also serve as references maps for future work to examine the potential spectrum of changes in gene expression that may occur throughout the growth phase in association with the cellular levels of ribosomes and rates of protein synthesis.

View larger version (105K): [in this window] [in a new window]   FIG. 3.   Two-dimensional gels of proteins from RB2256. (A and C) Logarithmic-phase (1.5 × 108 cells/ml) cells were labelled with [35S]methionine-cysteine for 60 min, and (B and D) cells starved for 24 h were labelled for 12 h. The molecular mass (in kilodaltons) of broad-range sodium dodecyl sulfate-PAGE standards (Bio-Rad) are indicated on the left of panel A. The molecular mass and pI values of specific spots were assigned with Melanie II software (Bio-Rad) and are shown in Table 3. Spots highlighted with circles and the letter L have 10-fold-higher intensities than the gels from logarithmic growth, and those with squares and the letter S have 10-fold-higher intensities than gels from starvation cultures. Spots identified with triangles and the letter U are present only in gels from logarithmic growth; some of the more intense spots are highlighted in the figure. Panels C and D are expanded views of sections of panels A and B, respectively, and serve to highlight the differences observable between protein spots from logarithmic-phase and starved cells.

View this table: [in this window] [in a new window]   TABLE 3.   Summary of protein characteristics for spots from two-dimensional protein gelsa that have spot intensities varying by 10-fold or more between logarithmic and stationary-phase cells

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

A comparison of gene expression and protein synthesis during growth and starvation in RB2256 and V. angustum S14. Differences in spot intensity of 60- to 70-fold are present between matching spots from protein gels for cells from logarithmic growth and starvation (Table 3), and 80 spots are present only in gels from logarithmic-phase cells (Table 2). These observed changes in spot intensity are indicative of growth phase-specific alterations in gene expression and provide strong support for the presence of a genetically programmed starvation response in RB2256.

1

1

1

1

1

1

1

1

Protein synthesis and ribosomes. We have previously shown that the ribosome content in RB2256 is highly regulated throughout the growth phase, and yet as cells can achieve maximum growth rates with a relative level of 10%, the cells appear to contain a cellular excess of ribosomes (11). To further examine the role of ribosomes in the cell, in this study we compared rates of protein synthesis and cellular protein content to the ribosome content (Fig. 1C). These data showed that the number of ribosomes per cell reached a maximum and then fell to basal levels approximately a half day before the rates of protein synthesis and cellular protein content reached maximum levels. In effect, the highest rate of protein translation occurs when there is a relative level of 10 to 15% of ribosomes (200 to 300 ribosomes cell¹). This indicates that ribosome synthesis is largely uncoupled from protein synthesis and that ribosomes may perform alternative functions during mid-logarithmic growth when the ribosome content greatly exceeds protein synthesis requirements. The ribosomes may function as protein reserves or be bound to and affect the stability of mRNA. These possibilities, however, need to be experimentally investigated.

Monitoring starvation and ecological relevance. In RB2256, maximum rates of RNA synthesis and protein synthesis coincide with the maximum rate of change in glucose concentration of the growth medium (Fig. 1). The rapid decrease in carbon flux during late logarithmic phase may provide a signal to the cell that starvation is imminent. In this way, much of this activity may be aimed at preparing the cell for complete nutrient deprivation.