surA, an Escherichia coli gene essential for survival in stationary phase.

Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation. The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur. The detailed analysis of one of these mutations is presented here. The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E. coli chromosome. It was located directly upstream of pdxA and formed part of a complex operon. Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype. Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase. Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml. Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population. The implications of these observations to our understanding of stationary-phase mutagenesis are discussed.     

Production and Repair of Radiochemical Damage in Escherichia coli Deoxyribonucleic Acid; Its Modification by Culture Conditions and Relation to Survival

Late log-phase Escherichia coli B/r cells are 1.6 times more sensitive to killing by X rays than are stationary-phase cells when grown in Brain Heart Infusion (BHI) + glucose. The number of single-chain breaks formed per krad is the same for log- and stationary-phase cells. Stationary-phase cells show a somewhat greater ability to repair single-chain breaks (especially after high doses of X rays) than do log-phase cells. The rapidity and extent of postirradiation deoxyribonucleic acid (DNA) degradation are greater in log-phase cells than in stationary-phase cells. The enhanced viability exhibited by stationary-phase cells thus appears to correlate both with enhanced single-chain break repair and the reduced degradation of DNA. Cells grown to stationary phase in peptone medium (PO cells) are 3.4 times more sensitive to killing by X rays than cells grown to stationary phase in peptone medium supplemented with glucose and phosphate buffer (PG cells). The yield of single-strand breaks is the same for both types of cells (but the absolute yield is about two times higher than in the cells grown in BHI + glucose). The kinetics for the repair of single-chain breaks are the same for both types of cells for about 30 min. After this time period, further repair ceases in the PO cells but continues in the PG cells, provided that glucose is present in the medium. Postirradiation DNA degradation is both more rapid and more extensive in PO cells than in PG cells whether or not glucose is present in the postirradiation incubation medium. The survival of stationary-phase E. coli B/r grown in PO or PG medium is likewise unaffected by the presence of glucose in the plating medium, and thus correlates better with the lower DNA degradation seen in the PG cells than with the increased strand rejoining, since this latter process requires the presence of glucose.     

The effect of thioredoxin on the radiosensitivity of bacteria

The ability of Escherichia coli thioredoxin to protect cells from lethal amounts of gamma radiation was tested using bacterial strains engineered to contain different amounts of thioredoxin per cell. Cells grown to late stationary phase demonstrated a decreasing sensitivity to gamma-radiation with increasing amounts of thioredoxin per cell. Exponentially growing cells were equally sensitive to the gamma-radiation regardless of the intracellular concentration of thioredoxin. Cells exhibiting the radiation-resistant phenotype in the stationary phase reverted to the radiation-sensitive phenotype when diluted into fresh growth medium. These results suggest that thioredoxin can protect cells from gamma-radiation under certain metabolic conditions.     

Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli

Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.     

Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent culture medium at high dilution

Abstract Micrococcus luteus starved for 2–7 months in spent medium following growth to stationary phase in batch culture exhibited a culturability (as estimated by direct plating on nutrient agar plates) of < 0.001%. However, following a lag, some 70% of the cells could be lysed upon inoculation into and cultivation in fresh lactate minimal medium containing penicillin, showing the capability of a significant portion of the cells at least to enlarge (and thus potentially to resuscitate). When the viable cell count was estimated using the most probable number method, by incubation of high dilutions of starved cells in liquid growth media, the number of culturable or resuscitable cells was very low, and little different from the viable cell count as assessed by plating on solid media. However, the apparent viability of these populations evidenced with the most probable number method was 1000–100 000-fold greater when samples were diluted into liquid media containing supernatants taken from the stationary phase of batch cultures of the organism, suggesting that viable cells can produce a factor which stimulates the resuscitation of dormant cells. Both approaches show, under conditions in which the growth of a limited number of viable cells during resuscitation is excluded, that a significant portion of the apparently non-viable cell population in an extended stationary phase is dormant, and not dead.     

The process of bacterial population splitting into dissociants and long-term batch cultivation of bacteria

The growth and composition of a population were studied during long-term (up to 50 days) batch cultivation of mono and mixed cultures of Pseudomonas aeruginosa S- and M-dissociants and Rhodobacter sphaeroides R- and M-dissociants without the addition of nutrients. During the cultivation of P. aeruginosa on a glucose-containing mineral medium, periodic lysis followed by polyculture growth resumption in the late stationary phase occurred on account of the M-dissociant: the change in its cell number corresponded to the change in the total cell number of the association. It was shown that the periodic occurrence of reducing sugars in the medium preceded the resumption of polyculture growth. Periodic secondary growth of the mixed culture of R. sphaeroides photosynthesizing bacteria occurred because of fast growing R-cells after the lysis of some part of the R-dissociant population. In the monoculture of the R. sphaeroides M-dissociant, R-cells were found during the whole period of cultivation, making up to 1–10% of the population irrespective of its size, which probably corresponded to the frequency of occurrence of this dissociant. In the R-dissociant monoculture, M-cells were found only after 26 days, and their number gradually decreased to half of population by the end of cultivation period. The joint growth of dissociants was characterized by the biomass increment and bacterial growth acceleration compared to monocultures, which is important for the fast development of new habitats under natural conditions. The cells of both bacterial species were lysed during long-term cultivation by exoproteinases secreted by the thin-wall cells of M-dissociants.     

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Continued growth of Escherichia coli after stopping medium addition to a K+-limited chemostat culture

The steady-state bacterial dry wt of Escherichia coli, growing under K+-limited conditions in the chemostat, was inversely dependent on the growth rate. This phenomenon was more carefully investigated in medium-flow stop experiments. Growth did not stop immediately but continued for a time, initially at the same rate as before. The dry wt increased to a value corresponding to a steady-state growth rate near zero, independent of the initial specific growth rate. This was observed in both the wild-type strain and a mutant that lacked the high-affinity K+ uptake system. The wild-type strain maintained a low extracellular K+ concentration both in the chemostat under steady-state conditions and after stopping the medium flow. The mutant, on the other hand, maintained a much higher extracellular K+ concentration in the steady state, which decreased to much lower values after stopping the medium flow. From the increase in bacterial dry wt and the low external K+ concentration after stopping the medium flow it is concluded that the intracellular K+ is redistributed among the cells, including new cells. The growth yield on K+ was highest in the stationary growth phase of a batch culture and all steady-state cultures converged ultimately to this yield value after the medium flow had been stopped. It is proposed that the growth rate of E. coli under K+-limited conditions is determined by the intracellular K+ concentration.     

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations

The RNA polymerase sigma factor, encoded by rpoS gene, controls the expression of a large number of genes in Escherichia coli under stress conditions. The present study investigated the growth characteristics and metabolic pathways of rpoS gene knockout mutant of E. coli growing in LB media under aerobic condition. The analyses were made based on gene expressions obtained by DNA microarray and RT-PCR, enzyme activities and intracellular metabolite concentrations at the exponential and early stationary phases of growth. Although the glucose utilization pattern of the mutant was similar to the parent strain, the mutant failed to utilize acetate throughout the cultivation period. Microarray data indicated that the expression levels of several important genes of acetate metabolism such as acs, aceAB, cysDEK, fadR, etc. were significantly altered in the absence of rpoS gene. Interestingly, there was an increased activity of TCA cycle during the exponential growth phase, which was gradually diminished at the onset of stationary phase. Moreover, rpoS mutation had profound effect on the expression of several other genes of E. coli metabolic pathways that were not described earlier. The changes in the gene expressions, enzyme activities and intracellular metabolite concentrations of the rpoS mutant are discussed in details with reference to the major metabolic pathways of E. coli.     

A gene between polA and glnA retards growth of Escherichia coli when present in multiple copies: physiological effects of the gene for spot 42 RNA.

We have isolated the single gene for spot 42 RNA of Escherichia coli on a 20-kilobase DNA fragment. Physical characterization of this cloned DNA fragment showed that it is homologous to a region at 86 min on the genetic map and extends from the 23S to 5S rRNA coding region of rrnA to the coding region of glnA, the gene for glutamine synthetase. Other genes included on this cloned DNA fragment are polA, ntrC (glnG), and ntrB (glnL). E coli cells transformed with a multicopy plasmid clone of the gene for spot 42 RNA had about a 10-fold increase in the amount of spot 42 RNA they contained. The amount of 6S RNA in these cells was increased about twofold, although the gene for 6S RNA was not located on this plasmid or on the larger 20-kilobase fragment. Presence of this multicopy plasmid also affected the growth of cells. The generation time was increased under a variety of growth conditions, especially when cells were grown in medium with succinate as the carbon source. In addition, some strains of E. coli which have multicopy plasmids carrying the gene for spot 42 RNA were unable to respond normally to a shift into richer medium: upon upshift from minimal glucose to LB broth or minimal glucose plus 1% Casamino Acids, there was a 3- to 4-h lag before the culture adapted to the new medium. More than 90% of the cells in such cultures stopped dividing, although they remained viable. The plating efficiency of minimal-glucose-grown cells was 100-fold less on rich media than on minimal glucose medium. One revertant was isolated which regained the phenotype of pBR322-transformed cells. Analysis of this strain showed that the plasmid it contained had an insertion of an IS1 element into the 5' end of the coding region for the gene for spot 42 RNA.     

Global Gene Expression Analysis of Long-Term Stationary Phase Effects in E. coli K12 MG1655

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.     

Culture Volume and Vessel Affect Long-Term Survival,Mutation Frequency,and Oxidative Stress of Escherichia coli

Bacteria such as Escherichia coli are frequently studied during exponential- and stationary-phase growth. However, many strains can survive in long-term stationary phase (LTSP), without the addition of nutrients, from days to several years. During LTSP, cells experience a variety of stressors, including reactive oxidative species, nutrient depletion, and metabolic toxin buildup, that lead to physiological responses and changes in genetic stability. In this study, we monitored survival during LTSP, as well as reporters of genetic and physiological change, to determine how the physical environment affects E. coli during long-term batch culture. We demonstrate differences in yield during LTSP in cells incubated in LB medium in test tubes versus Erlenmeyer flasks, as well as growth in different volumes of medium. We determined that these differences are only partially due to differences in oxygen levels by incubating the cells in different volumes of media under anaerobic conditions. Since we hypothesized that differences in long-term survival are the result of changes in physiological outputs during the late log and early stationary phases, we monitored alkalization, mutation frequency, oxidative stress response, and glycation. Although initial cell yields are essentially equivalent under each condition tested, physiological responses vary greatly in response to culture environment. Incubation in lower-volume cultures leads to higher oxyR expression but lower mutation frequency and glycation levels, whereas incubation in high-volume cultures has the opposite effect. We show here that even under commonly used experimental conditions that are frequently treated as equivalent, the stresses experienced by cells can differ greatly, suggesting that culture vessel and incubation conditions should be carefully considered in the planning or analysis of experiments.     

Sensitivity of Escherichia coli cells to seawater closely depends on their growth stage.

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.     

Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response

When Campylobacter jejuni NCTC 11351 was grown microaerobically in rich medium at 39 degrees C, entry into stationary phase was followed by a rapid decline in viable numbers to leave a residual population of 1% of the maximum number or less. Loss of viability was preceded by sublethal injury, which was seen as a loss of the ability to grow on media containing 0.1% sodium deoxycholate or 1% sodium chloride. Resistance of cells to mild heat stress (50 degrees C) or aeration was greatest in exponential phase and declined during early stationary phase. These results show that C. jejuni does not mount the normal phenotypic stationary-phase response which results in enhanced stress resistance. This conclusion is consistent with the absence of rpoS homologues in the recently reported genome sequence of this species and their probable absence from strain NCTC 11351. During prolonged incubation of C. jejuni NCTC 11351 in stationary phase, an unusual pattern of decreasing and increasing heat resistance was observed that coincided with fluctuations in the viable count. During stationary phase of Campylobacter coli UA585, nonmotile variants and those with impaired ability to form coccoid cells were isolated at high frequency. Taken together, these observations suggest that stationary-phase cultures of campylobacters are dynamic populations and that this may be a strategy to promote survival in at least some strains. Investigation of two spontaneously arising variants (NM3 and SC4) of C. coli UA585 showed that a reduced ability to form coccoid cells did not affect survival under nongrowth conditions.     

Transition from positive to neutral in mutation fixation along with continuing rising fitness in thermal adaptive evolution

It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2°C or 4°C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9°C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.     

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.