Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp. niger.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1. Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells. The effect of growth rate was studied by varying the dilution rate. However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited. The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content. No indications were obtained that the composition of the peptidoglycan of B. subtilis subsp. niger WM was phenotypically variable.     

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.     

Alterations in lipopolysaccharide produced by chemostat-grown Escherichia coli O157:H7 as a function of growth rate and growth-limiting nutrient

Escherichia coli O157:H7 was grown in chemostats as continuous cultures at different controlled growth rates and under different nutrient limitations to determine the effects on lipopolysaccharide (LPS) structure. LPS from whole cells and extracted using the hot aqueous phenol method was examined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration after hydrolysis with acetic acid. At low growth rates under glucose limitation (D = 0.1 h-1, doubling time (td), approx. 416 min; or D = 0.4 h-1, td, approx. 104 min), E. coli O157 produced high molecular weight LPS identical to that previously characterized from cells grown in batch culture. At a high growth rate (D = 0.8 h-1, td, approx. 52 min), the ratio of high molecular weight LPS to low molecular weight LPS produced greatly decreased. A small amount of high molecular weight LPS, containing O-polysaccharide which lacked amino sugars, and which thus was chemically different from that previously characterized, was produced by the cells at high growth rates. The predominant form of LPS from these cells was of slightly higher molecular weight than rough LPS, probably S-R LPS, and it consistently formed aggregates on SDS-PAGE. This form of LPS was also predominant when E. coli O157 was grown under Mg2+ limitation at an intermediate growth rate (D = 0.4 h-1, td, approx. 104 min).     

Effect of Water Stress on Cortical Cell Division Rates within the Apical Meristem of Primary Roots of Maize

We characterized the effect of water stress on cell division rates within the meristem of the primary root of maize (Zea mays L.) seedlings. As usual in growth kinematics, cell number density is found by counting the number of cells per small unit length of the root; growth velocity is the rate of displacement of a cellular particle found at a given distance from the apex; and the cell flux, representing the rate at which cells are moving past a spatial point, is defined as the product of velocity and cell number density. The local cell division rate is estimated by summing the derivative of cell density with respect to time, and the derivative of the cell flux with respect to distance. Relatively long (2-h) intervals were required for time-lapse photography to resolve growth velocity within the meristem. Water stress caused meristematic cells to be longer and reduced the rates of cell division, per unit length of tissue and per cell, throughout most of the meristem. Peak cell division rate was 8.2 cells mm-1 h-1 (0.10 cells cell-1 h-1) at 0.8 mm from the apex for cells under water stress, compared with 13 cells mm-1 h-1 (0.14 cells cell-1 h-1) at 1.0 mm for controls.     

Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa.

An in vitro chemostat system was used to study the growth and the expression of iron-regulated outer-membrane proteins (IROMPs) by biofilm cells of Pseudomonas aeruginosa cultivated under conditions of iron limitation. The population of the planktonic cells decreased when the dilution rate was increased. At a dilution rate of 0.05 h-1, the populations of planktonic cells of both mucoid and nonmucoid P. aeruginosa were 3 x 10(9) cells/mL. This value dropped to 5 x 10(6) cells/mL when the dilution rate was increased to 1.0 h-1. The reverse was observed for the biofilm cells. The number of biofilm cells colonising the silicone tubing increased when the dilution rate was increased. The number of biofilm cells of the mucoid strain at steady state was 2 x 10(8) cells/cm (length) when the dilution rate was fixed at 0.05 h-1. The figure increased to 8 x 10(9) cells/cm when the dilution rate was increased to 1.0 h-1. The population of biofilm cells of the nonmucoid strain was 9 x 10(7) cells/cm (length) when the dilution rate was 0.05 h-1. It increased to 2 x 10(9) cells/cm when the dilution rate was set at 1.0 h-1. The expression of IROMPs was induced in the biofilm cells of both mucoid and nonmucoid strains when the dilution rates were 0.05 and 0.2 h-1. IROMPs were reduced but still detectable at the dilution rate of 0.5 h-1. However, the expression of IROMPs was repressed when the dilution rate was increased to 1.0 h-1. The data suggest that the biofilm cells of P. aeruginosa switch on the expression of IROMPs to assist iron acquisition when the dilution rate used for the chemostat run is below 0.5 h-1. The high affinity iron uptake system is not required by the biofilm cells when the dilution rate is increased because the trace amount of iron present in the chemostat is sufficient for the growth of adherent biofilm cells.     

Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic- or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 microM, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

Changes in peptidoglycan composition and penicillin-binding proteins in slowly growing Escherichia coli.

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Stability of TOL plasmid pWW0 in Pseudomonas putida mt-2 under non-selective conditions in continuous culture

Pseudomonas putida mt-2, harbouring the TOL plasmid pWW0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. The fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (TOL- cells), was determined. Genetic analysis revealed that all TOL- cells isolated harboured partially deleted plasmids, lacking the TOL catabolic genes. The growth-rate advantage of the TOL- cells was quantified from the kinetics of their increase as a fraction of the total population. At a dilution rate of 0.1 h-1 no growth-rate advantage of TOL- cells was found when phosphate or ammonium were limiting. Under sulphate-limitation, ingrowth of TOL- cells was evident but did not follow a straightforward pattern. Under succinate-limitation the growth-rate advantage was the highest, particularly at low dilution rates (about 50% at D = 0.05 h-1). In phauxostat culture, at the maximal growth rate, the growth-rate advantage of TOL- cells was less than 1%. The specific activity in TOL+ cells of the plasmid-encoded enzyme catechol 2,3-dioxygenase was relatively high at a low growth rate.     

Production of the fimbrial adhesin 987P by enterotoxigenic Escherichia coli during growth under controlled conditions in a chemostat

The effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (mu). Under aerobic growth conditions fimbriae production increased with higher mu values till mu = 0.40 h-1 and decreased again at mu values close to mu max (0.48 h-1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to mu max (0.16 h-1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of mu. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+ cell and a slower reduction in the percentage of P+ cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+ cells and a constant amount of fimbriae per P+ cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+----P- switch between two steady states was 2.7 x 10(-2). In batch culture the frequency of the P(-)----P+ switch was minimally 2.9 x 10(-2). The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.     

Improvement of Y-values of Hansenula polymorpha growth on methanol by simultaneous utilization of glucose

The simultaneous utilization of methanol and glucose by Hansenula polymorpha MH20 was investigated in chemostat (C-limited) cultivation. The mixed-substrate utilization results in biomass yields which are greater up to 20 to 25% as expected assuming an additive growth on both substrates. This is referred to as an auxiliary-substrate effect. Additionally, methanol can be utilized at higher growth rates in the presence of glucose compared to those obtained on this substrate alone. The extend of the auxiliary-substrate effect and the optimum ratio of substrates to reach this effect depend on dilution rate. The greatest stimulation in yield is obtained at D approximately 0.1 h-1, after raising the dilution rate this effect diminishes. At a rate of 0.1 h-1 the optimum mixed-substrate ratio of methanol: glucose is 7:1 (g). By increasing the growth rate the ratio changes toward glucose and reached a value of 1:1 (g) at D = 0.3 h-1. This change in the optimum ratio correlates with diminution in yield coefficient of methanol accompanying an increase in growth rate greater than 0.15 h-1. Energy balances of the utilization of the single substrates are used for interpretation of these results. From this it is evident that methanol does not play the role of an energy-rich substrate in the metabolism of yeast. Rather glucose is the energy-providing substrate in this combination.     

Response of Rhodopseudomonas capsulata to illumination and growth rate in a light-limited continuous culture.

Rhodopseudomonas capsulata was grown under anaerobic, photosynthetic conditions in a continuous culture device. Under light limitation, at a constant dilution rate, it was shown that cell composition, including photopigment (bacteriochlorophyll and carotenoids) and ribonucleic acid content, was not affected by incident light intensity; however, steady state culture density varied directly and linearly with light intensity. On the other hand, photopigment and ribonucleic acid levels were affected by growth rate regardless of light intensity. Additional experiments indicated a high apparent Ks for growth of R. capsulata with respect to light. These results were interpreted to mean that near the maximum growth rate (D = 0.45 h-1) some internal metabolic process became the limiting factor for growth, rather than the imposed energy limitation. A mathematical expression for the relation between steady-state culture density and dilution rate was derived and was found to adequately describe the data. A strong correlation was found between continuous cultures limited either by light or by a chemical energy source.     

Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of Klebsiella pneumoniae

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.     

Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment

A Spirillum sp. and a Pseudomonas sp. possessing crossing substrate saturation curves for L-lactate were isolated from fresh water by chemostat enrichment. Their Ks and mumax values for L-lactate were: Spirillum sp., 23 micrometer and 0.35 h-1, respectively; Pseudomonas sp., 91 micrometer and 0.64 h-1, respectively. Under L-lactate limitation, pseudomonas sp. outgrew Spirillum s. at dilution rates (D) above 0.29 h-1, but the converse occurred at lower D values. The advantage of Spirillum sp. increased with decreasing D until, at D = 0.05 h-1 (i.e. L-lactate concentration of approximately 1 micrometer), Pseudomonas sp. was eliminated from the culture essentially as a non-growing population. In Spirillum sp. the Km for L-lactate transport (5.8 micrometer) was threefold lower than in Pseudomonas sp. (20 micrometer); Spirillum sp. also possessed a higher Vmax for the transport of this substrate. The surface to volume ratio was higher in Spirillum sp. and increased more markedly than in Pseudomonas sp. in response to decreasing D. Thus, a more efficient scavenging capacity contributes to the advantage of Spirillum sp. at low concentrations of the carbon source. Although most of the enzymes of L-lactate catabolism were more active in Pseudomonas sp., NADH oxidase activity was about twice as high in Spirillum sp.; and, unlike Pseudomonas sp., the cytochrome c content of this bacterium increased markedly with decreasing D. A more active and/or more efficient respiratory chain may therefore also play a role in the advantage of Spirillum sp. The other factors which appear to be involved include a lower energy of maintenance of Spirillum sp. [0.016 g L-lactate (g cell dry wt)-1 h-1 compared with 0.066 in Pseudomonas sp.] and a lower minimal growth rate.     

Kinetics of uptake and incorporation of meso-diaminopimelic acid in different Escherichia coli strains.

The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments. For different E. coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell. With E. coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time. This lag period can be ascribed to the presence of a large internal DAP pool in the cells. An E. coli K-12 lysA strain was constructed which could be grown without DAP in its medium. Consequently, due to the higher specific activity of the added [3H]DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain. In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool. The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine. These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis. To explain our observations, we suggest the existence of an expandable pool of DAP in E. coli which varies with the DAP concentration in the growth medium. With 2 microgram of DAP per ml, the size of the pool is severalfold the amount of DAP contained in the cell wall. This pool can be partly washed out of the cells. Grown without DAP, MC4100 lysA still has a small pool caused by endogenous synthesis, which accounts for the fact that steady-state [3H]DAP incorporation in the lysA strain still shows a lag period.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.