Effects of mutations in the rpoS gene on cell viability and global gene expression under nitrogen starvation in Escherichia coli

Md. Shahinur Kabir1, Takehiro Sagara1, Taku Oshima2,3, Yuya Kawagoe3, Hirotada Mori2,3, Ryouichi Tsunedomi1 and Mamoru Yamada1

1 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
2 CREST, Nara Institute of Science and Technology, Nara 630-01, Japan
3 R&E Center of Informatics, Nara Institute of Science and Technology, Nara 630-01, Japan

Correspondence
Mamoru Yamada
m-yamada{at}yamaguchi-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Escherichia coli bearing an rpoS amber or disrupted mutation exhibited a significant decrease in the number of colony-forming units (c.f.u.) when exposed to nitrogen starvation, which was not observed in cells bearing a functional rpoS allele. The decrease in the number of c.f.u. that was observed about 25 h after initiation of nitrogen starvation was prevented by the addition of nitrogen within 3 h but not by the addition of nitrogen at more than 7 h after the initiation of nitrogen starvation, suggesting that a process leading to a decline in c.f.u. starts within this period. DNA microarray analysis of the rpoS mutant showed that a large number of genes including many functionally undefined genes were affected by nitrogen starvation. The expression levels of {sigma}S and {sigma}H regulon genes encoding acid-resistant proteins (hdeA, hdeB, gadA and gadB), DNA-binding protein (dps), chaperones (dnaK, ibpA, ibpB, dnaJ and htpG), chaperonins (mopB and mopA) and energy-metabolism-related proteins (hyaABCDF and gapA), and those of other genes encoding nucleotide-metabolism-related proteins (deoC and deoB), cell-division protein (ftsL), outer-membrane lipoprotein (slp) and DNA-binding protein (stpA) were significantly decreased by 10 h nitrogen starvation. The genes encoding transport/binding proteins (nac, amtB, argT, artJ, potF and hisJ) and amino acid-metabolism-related proteins (glnA, trpB, argG, asnB, argC, gdhA, cstC, ntrB, asd and lysC) were significantly up-regulated under the same condition, some of which are known Ntr genes expressed under nitrogen limitation. On the basis of these results, possible causes of the decrease in the number of c.f.u. under nitrogen starvation are discussed.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Escherichia coli has a life cycle in which exponential growth occurs in the presence of sufficient nutrients but ceases as nutrients are consumed or as growth-suppressing factors accumulate; at the same time, cells develop resistance to a variety of environmental stresses (Hengge-Aronis, 1993; Kolter et al., 1993; McCann et al., 1991; Siegele & Kolter, 1992). The {sigma}S subunit, encoded by rpoS, of RNA polymerase, which functions as a master regulator of general stress responses, seems to be involved in the cessation of growth and in the acquisition of resistance (Hengge-Aronis, 1996; Ishihama, 1997; McCann et al., 1991; Muffler et al., 1997). The rpoS gene is required for prolonged survival in the stationary phase in both nutrient-rich and minimal media (Lange & Hengge-Aronis, 1991a), for stationary-phase-induced acid resistance (Small et al., 1994), for thermotolerance (Hengge-Aronis et al., 1991; Muffler et al., 1997) and for adaptation to growth in a medium of high osmolarity (Hengge-Aronis, 1996; Hengge-Aronis et al., 1993). While the {sigma}S level is low in rapidly growing cells not exposed to any stress, it increases in response to environmental stresses (Hengge-Aronis et al., 1991; Ishihama, 1997; Zambrano & Kolter, 1996).

E. coli exhibits a large decrease, by one to two orders of magnitude, in the number of colony-forming units (c.f.u.) in early stationary phase and then retains the same level of viability for many days (Kolter et al., 1993; Zambrano et al., 1993). A number of genes are thought to be involved in the decline of cell viability. The ssnA gene, which is under the negative control of {sigma}S has been shown to enhance the decline (Talukder et al., 1996; Yamada et al., 1999). A proportion of cells unable to form colonies appears to lyse via the regulon of {sigma}E (Nitta et al., 2000), a factor controlling the expression of genes encoding extracytoplasmic proteins (De Las Peñas et al., 1997; Missiakas & Raina, 1997). Nutrients derived from the lysed cells could allow some cells to grow in the following phase. However, most of the molecular mechanisms underlying these phenomena have not been elucidated.

Nutrient starvation seems to be one of the factors causing a decline in the number of c.f.u. in early stationary phase. Experiments on glucose starvation have given contradictory results: no significant difference was found between viability in glucose-starved conditions and that in non-starved conditions over a period of 1 week in some studies (Lange & Hengge-Aronis, 1991b), while a significant loss of viability in nearly the same period was observed in other studies (Davis et al., 1986; Reeve et al., 1984). This discrepancy could be due to differences in strain backgrounds. Mutation in rpoS has been reported to enhance the decrease in the number of c.f.u. under nutrient-starved conditions in some Gram-negative bacteria (Fang et al., 1992; Hengge-Aronis, 1993; Jorgensen et al., 1999; McCann et al., 1991; O'Neal et al., 1994), and a large number of genes appear to be under the control of {sigma}S in E. coli (Hengge-Aronis, 1996; Ishihama, 1999; Matin, 1991). An E. coli rpoS-defective mutant was demonstrated to cause a pleiotropic phenotype, exhibiting sensitivity to H2O2, low level of acid phosphatase, deficiencies in glycogen synthesis as a storage compound, and alteration in cell size and shape in the stationary phase (Hengge-Aronis & Fischer, 1992; Lange & Hengge-Aronis, 1991b; Loewen & Triggs, 1984; Touati et al., 1986). Even in a nutrient-rich medium, the decline in the number of c.f.u. in early stationary phase increased in the rpoS mutant (Yamada et al., 1999).

Limitation of the nitrogen source below a certain level induces the NtrB/NtrC/{sigma}N regulatory system (Hengge-Aronis, 1993) that allows E. coli to establish high-affinity uptake systems for nitrogen sources, scavenging nitrogen by inducing Ntr genes encoding transport proteins, amtB, argT-hisJMPQ, codB, cycA, ddpXABCDE, gabP, glnHPQ, gltIJKL, nupC, oppABCD and yhdWXYZ (Zimmer et al., 2000). Under nitrogen starvation, the signal transduction by GlnK, encoded by the glnK gene, may be crucial for survival and also for the expression of some Ntr genes (Blauwkamp & Ninfa, 2002). However, there are few reports on the effect of nitrogen starvation on E. coli global gene expression.

In this study, an rpoS-deficiency-specific decline in c.f.u. under nitrogen starvation was shown by disruption of the rpoS gene and complementation with a functional rpoS gene. We then focused on the identification of the genes influenced by the nitrogen starvation in an rpoS amber mutant, a predominantly occurring mutant in E. coli K-12 (Atlung et al., 2002). This experiment was performed by DNA microarray to follow the change of global gene expression as nitrogen starvation proceeded. Some of the results were confirmed by RT-PCR.


   METHODS
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Materials.
Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo and New England Biolabs. DNA sequencing kits were obtained from Applied Biosystems Japan. Oligonucleotide primers were synthesized by Proligo Japan K.K. Other chemicals were all of analytical grade.

Bacterial strains, plasmids and culture media.
The bacterial strains used in this study were derivatives of E. coli K-12. Their relevant genotypes and plasmids are presented in Table 1. Cell culture was performed using modified Luria–Bertani (LB) medium [1 % (w/v) Bactotryptone, 0·5 % (w/v) yeast extract and 0·5 % (w/v) NaCl] or M9 minimal medium containing 0·2 % (w/v) glucose (Miller, 1992) at 37 °C under aerobic conditions by reciprocal shaking (100 times min–1). Antibiotics were added as appropriate, at the following final concentrations: 50 µg ampicillin ml–1, 8 µg tetracycline ml–1. The mutation of rpoS : : Tn10 was transferred from UM122 by P1 transduction (Miller, 1992).


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Table 1. Bacterial strains and plasmids

 
Starvation experiments.
A single colony was inoculated into 3 ml M9 minimal medium for 16 h. The pre-cultured cells were transferred into M9 minimal medium to dilute to a turbidity corresponding to an OD600 of 0·1, and were grown for 24 h (early stationary phase). The cells were recovered as a pellet by low-speed centrifugation and washed with M9 minimal medium lacking NH4Cl for nitrogen starvation. The cells were resuspended in the same medium lacking the nitrogen source and subjected to starvation under aerobic conditions. Cell viability was determined by counting the number of c.f.u. on modified LB agar plates. The plates were incubated at 37 °C and the colonies formed on the plates were counted after 24 h. The experiment was independently conducted at least three times.

Cloning and analysis of the rpoS genes from W3110Y and W3110N.
Conventional recombinant DNA techniques (Izu et al., 1997; Sambrook et al., 1989) were applied. The rpoS gene from W3110Y or W3110N was cloned after PCR amplification as described previously (Yamada et al., 1993) using two primers, 5'-AATCCGTAAACCCGCTG-3' and 5'-CTTAATTACCTGGTGCG-3', and each corresponding genomic DNA as a template. An amplified 1·2 kb DNA fragment was inserted into the multi-cloning site of the pGEM-T vector and then subjected to nucleotide sequencing (Sanger et al., 1977).

Construction of an expression plasmid of the rpoS gene from W3110N.
A 1·0 kb DNA fragment bearing rpoS was amplified by PCR using primers 5'-TACGGATCCAGTCAGAATACGCTGAA-3' and 5'-TACAAGCTTTCATTACTCGCGGAACAGCG-3', with BamHI and HindIII sites, respectively, at their 5'-ends, and W3110N genomic DNA as a template. The amplified DNA fragment was digested with BamHI and HindIII and inserted into the multi-cloning site of pQE-80L, generating pQERPOS. The identity of the inserted fragment was confirmed by nucleotide sequencing. Cells harbouring pQERPOS were grown at 37 °C in M9 minimal medium containing ampicillin, and the promoter was induced by the addition of 0·1 mM IPTG.

Preparation of RNA for DNA microarray.
W3110Y cells were cultured and subjected to nitrogen starvation as described above. Cells that had been grown in 30 ml M9 minimal medium with or without nitrogen were harvested after 3 h, 7 h and 10 h incubation under starvation conditions, and total RNA was immediately prepared according to the hot phenol method (Aiba et al., 1981). After phenol/chloroform treatment and ethanol precipitation, the resultant RNA (about 100 µg) was resuspended in 100 mM NaOAc, pH 5·5, 50 mM MgSO4 and treated at 37 °C for 1 h with 10 units of RNase-free DNase (Takara Shuzo) in a final volume of 100 µl. RNA was then recovered after phenol/chloroform treatment and ethanol precipitation. DNA microarray preparation, fluorescent-labelled cDNA preparation, array hybridization, and data capturing and analysis were performed as described previously (Oshima et al., 2002). Two independently obtained mRNA preparations of the cells at each starvation period (3 h, 7 h and 10 h) were examined, and each preparation was subjected twice to microarray analysis. We thus obtained four independent data sets per gene per starvation period. The consistency of the different expressions in the four data sets obtained was calculated using the Wilcoxon signed rank test on the PYTHON program. Thus, spots with a significantly (P<0·01) lower (<0·5, i.e. a negative fold difference) or higher (>2, i.e. a positive fold difference) fluorescent ratio of the nitrogen-starved sample to the control sample were considered to be real differences as described previously (Oshima et al., 2002). The values shown in Tables 4 and 5 are the means of the four independently obtained data for each gene. Functional classification of genes was performed according to EcoCyc (Karp et al., 2002) and KEGG (Kanehisa et al., 2002) databases and classifications of {sigma}S regulon, {sigma}H regulon and {sigma}N regulon were done based on Ishihama (2000), Gross (1996) and Magasanik (1996), respectively.


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Table 4. {sigma}S and {sigma}H regulon genes significantly affected by nitrogen starvation in W3110Y

Total RNAs were prepared from cells exposed to 3 h, 7 h or 10 h nitrogen starvation and subjected to DNA microarray analysis as described in Methods.

 

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Table 5. Effect of nitrogen starvation on gene expression in different E. coli strains

W3110Y, W3110N and YU635 (W3110N rpoS : : Tn10) were grown in M9 minimal medium and then either nitrogen-starved or not for 10 h. Total RNAs were then prepared from the cells and subjected to RT-PCR analysis. Procedures of these processes and estimation of the RT-PCR products are described in Methods. The ratio was calculated by comparing amount of the RT-PCR products obtained from nitrogen-starved (–N) cells and that from non-starved (+N) cells.

 
RT-PCR analysis.
RT-PCR analysis was performed using an mRNA Selective RT-PCR Kit (Takara Shuzo) to examine the expression of several genes. The primer sets used are listed in Table 2. Total RNA was prepared from 10 h nitrogen-starved W3110Y, W3110N or YU635 (W3110N rpoS : : Tn10) cells as described above. The RT reaction was carried out at 50 °C for 15 min with 0·1 µg of total RNA and each downstream primer, and subsequently, PCR consisting of denaturing at 85 °C for 1 min, annealing at 45 °C for 1 min and extension at 72 °C for 1 min was carried out using the two primers for each gene. The PCR products after 20, 25, 30 and 35 cycles were analysed by 0·9 % (w/v) agarose gel electrophoresis and stained with ethidium bromide. As a control, 10 µg total RNA was run on 1·2 % (w/v) agarose gel electrophoresis followed by staining with ethidium bromide to make sure that equivalent amounts of RNAs were used between nitrogen-starved samples and non-starved samples. The relative amounts of RT-PCR products on the gel were compared by measuring band density after the colour of the image taken had been reversed by using model GS-700 Imaging Densitometer (Bio-Rad). Linearity of the amplification was observed at least up to the 35th cycle. In our conditions, the RNA-selective RT-PCR was able to specifically detect mRNA because no band was observed when reverse transcriptase was omitted.


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Table 2. Primers used for RT-PCR in this study

 

   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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rpoS mutation and reduction in the number of c.f.u. under nitrogen-starved conditions
To elucidate the mechanism by which E. coli undergoes a large decrease in viable cell number in early stationary phase, we examined the effect of nitrogen starvation on survival with cells grown to the early stationary phase. W3110Y, one lineage of strain W3110, was grown for 24 h in minimal medium, corresponding to the early stationary phase, and then subjected to nitrogen starvation as shown in Fig. 1(a). The number of c.f.u. decreased by about two orders of magnitude after 150 h under nitrogen-starved conditions. No significant decrease in OD600 between starved and non-starved conditions, however, was observed. On the other hand, W3110N, another strain, showed no such reduction in the number of c.f.u. under the same conditions of nitrogen starvation (see Fig. 2a).



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Fig. 1. Effect of nitrogen starvation (a) and nitrogen concentration (b) on c.f.u. in W3110Y. (a) Cells were grown in M9 minimal medium at 37 °C for 24 h. The cells were recovered as a pellet by centrifugation, suspended (time zero) in M9 minimal medium containing nitrogen source ({blacksquare}) or lacking nitrogen source ({square}), and further incubated at 37 °C. (b) W3110Y cells were grown and recovered as a pellet as described in (a) but suspended in M9 minimal medium containing different concentrations of nitrogen source, 18·7 mM ({blacksquare}), 6·2 mM ({circ}), 0·62 mM ({blacktriangleup}) or 0 mM ({square}), and further incubated at 37 °C. In one experiment, cells were suspended in M9 minimal medium containing 18·7 mM NaCl ({bullet}) instead of NH4Cl. Aliquots were removed from the culture at times indicated and plated on modified LB agar plates. Colonies were counted after incubation at 37 °C for 24 h. These experiments were performed at least three times. The error bars represent the standard deviation from the mean.

 


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Fig. 2. Protection of c.f.u. decline by the rpoS gene under nitrogen-starved condition. (a) W3110N cells ({blacksquare}, {square}) or YU635 (W3110N rpoS : : Tn10) cells ({bullet}, {circ}) were grown and recovered as described in Fig. 1. Cells were then suspended in M9 minimal medium containing nitrogen source (closed symbols) or lacking nitrogen source (open symbols), and further incubated at 37 °C. (b) YU635 cells harbouring pQERPOS ({blacksquare}, {square}) or the vector pQE-80L ({bullet}, {circ}) were grown and recovered as described in Fig. 1. Cells were suspended in M9 minimal medium containing nitrogen source (filled symbols) and lacking nitrogen source (open symbols), and further incubated at 37 °C. C.f.u. were determined as described in Fig. 1. Ampicillin (50 µg ml–1) was added when needed. The experiments were performed at least three times. The error bars represent the standard deviation from the mean.

 
The effect of nitrogen concentration on W3110Y c.f.u. was then examined (Fig. 1b). In comparison with the standard nitrogen concentration (18·7 mM NH4Cl) in minimal medium, 0·62 mM NH4Cl seemed insufficient for the maintenance of c.f.u. Notably, the NH4Cl-depletion effect did not appear to be due to the osmotic effect because an equimolar amount of NaCl to the standard amount of NH4Cl could not maintain the level of c.f.u. in the absence of the standard NH4Cl. The decline in the number of c.f.u. was not prevented even by the addition of 30 mM Mg2+ (data not shown). Therefore, it is likely that nitrogen is important for the maintenance of W3110Y c.f.u.

Such sensitiveness of W3110Y to low nitrogen concentration led us to suspect mutations in rpoS, encoding a stress sigma factor. The nucleotide sequences of the rpoS genes in strains W3110Y and W3110N were determined after cloning the entire genes by PCR-based amplification. Only one nucleotide difference was found between the rpoS genes of the two strains: W3110N possesses the same nucleotide sequence as that deposited in genomic sequence databases, whereas W3110Y has an amber mutation (TAG) at the 33rd codon.

Amber mutation at the 33rd codon of rpoS has been found in more than 50 % of the original E. coli K-12 isolates, and has been demonstrated to occur at a hot spot; and the evolution of the 33rd codon from the amber mutation to glutamine codon (CAG), as in the case of W3110N, has been proposed (Atlung et al., 2002). Other E. coli K-12 strains, FB8 (from P. Postma) and G6 (from T. Ferenci), sensitive and resistant to nitrogen starvation were found to have the same nucleotide sequences as W3110Y and W3110N, respectively (unpublished). Therefore, the amber mutation at the 33rd codon seems to occur commonly in E. coli K-12. Additionally, several mutations of rpoS were found in different stocks of E. coli (Jishage & Ishihama, 1997). Thus, such rpoS mutations would provide growth advantages under experimental conditions or during storage. A recent study has suggested that one of the physiological advantages of rpoS mutation is enhanced production of scavenger transporters due to the low level of RpoS protein molecules (Notley-McRobb et al., 2002). We also observed a survival advantage in that W3110Y and YU635 (W3110N rpoS : : Tn10) were less sensitive to short-term carbon starvation than W3110N (unpublished).

Involvement of the {sigma}S regulon in maintenance of c.f.u. level under nitrogen-starved conditions
To further examine the relationship between the rpoS mutation and nitrogen-starvation-dependent reduction in the number of c.f.u., YU635 (W3110N rpoS : : Tn10) was subjected to nitrogen starvation. A large reduction in the number of c.f.u. was observed in comparison to that in the parental strain as shown in Fig. 2(a). Further experiments using the functional rpoS plasmid clone from W3110N were performed. Introduction of the plasmid resulted in a significant increase in the number of c.f.u. of YU635 compared to that in the case of the vector plasmid (Fig. 2b). The reduction in the number of c.f.u. in W3110Y was also prevented to a considerable degree by the rpoS clone. Moreover, the pattern of decline in the number of c.f.u. of W3110Y was found to be similar to that of YU635 and also to that of an rpoS : : Tn10 derivative from W3110Y in minimal medium lacking nitrogen (data not shown). Taken together, the results suggest that the rpoS mutations directly cause the reduction in the number of c.f.u. under nitrogen-starved conditions, and thus that the {sigma}S regulon genes perform crucial functions in the maintenance of viable cell number under the conditions.

Effect of duration of nitrogen starvation on c.f.u.
To examine whether the reduction in the number of c.f.u. was fated once nitrogen had been eliminated from the medium, a sufficient amount of NH4Cl was added to nitrogen-starved culture media 1–23 h after the starvation had been initiated (Fig. 3). W3110Y was tested since it was a predominantly occurring rpoS mutant strain. No significant reduction in the number of c.f.u. was observed during the first 3 h of the nitrogen-starvation period. However, when the starvation was continued for 23 h, the c.f.u. reduction was no longer suppressed by the addition of nitrogen and the reduction level was almost the same as that of the control without nitrogen. Starvation for 7–10 h had a moderate effect on c.f.u. These results suggest that nitrogen starvation for a short period may be transient and reversible, but that further nitrogen starvation causes cells to enter an irreversible process leading to the reduction in the number of c.f.u. Notably, although the c.f.u. level after 23 h nitrogen starvation was not greatly different from that at the initiation of nitrogen starvation, the supply of nitrogen to a 23 h nitrogen-starvation culture was not able to suppress the reduction in the number of c.f.u. Therefore, we assume that a cell death process is induced by nitrogen depletion in E. coli rpoS mutants and that the process leading to cell death may be unavoidable once the initial trigger has been established. Alternatively, such cell death could be due to the depletion of some essential metabolites or pathway upon prolonged nitrogen starvation.



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Fig. 3. Effect of duration of nitrogen starvation on c.f.u. in W3110Y. W3110Y cells were grown and recovered as described in Fig. 1. Cells were then suspended in M9 minimal medium lacking nitrogen source and incubated at 37 °C for 0 h ({blacksquare}), 1 h ({triangleup}), 3 h ({circ}), 7 h ({bullet}), 10 h ({lozenge}), 23 h ({blacktriangleup}) or 120 h ({square}). Successively, nitrogen source was added to each culture and further incubation was carried out for a total of 120 h. c.f.u. was determined as described in Fig. 1. The experiments were performed three times. The error bars represent the standard deviation from the mean.

 
Starvation for more than 3 h might result in exhaustion of the endogenous nitrogen source, which would have a drastic effect on the rpoS mutant cells, inducing an irreversible process leading to cell death. The functional rpoS and its regulon, on the other hand, are assumed to somehow direct an appropriate adjustment of cellular activities, including replication, transcription and translation, as is expected to occur in stationary phase, enabling cells to survive under nitrogen-starved conditions.

Change in gene expression following elimination of nitrogen from the medium
The results presented above prompted us to examine the change in global gene expression in the rpoS mutation background as nitrogen starvation proceeded by using a DNA microarray including most E. coli genes. For preparing the probes, on the basis of the results shown in Fig. 3, W3110Y cells were exposed to nitrogen starvation in a minimal medium lacking nitrogen and total RNA was isolated from cells that had been starved for 3 h, 7 h and 10 h. The RNA for control probes was prepared from cells of the same strain that had been grown in parallel in a replete minimal medium. The genes whose expression was significantly affected by the starvation were defined as those that exhibited a fluorescent intensity more than twice the than that of the control or less than 50 % of that of the control (Oshima et al., 2002), and such genes were classified into induced genes and repressed genes, respectively, as shown in Table 3. Under the condition of 3 h nitrogen starvation, 8–184-fold increases in expression level were observed in glnH, glnA, argT and nac genes, known as the {sigma}N regulon, and 5–70- and 2–87-fold increases were observed in the same genes in cells exposed to 7 h and 10 h of starvation, respectively, indicating that cells indeed seemed to be nitrogen-starved under the conditions used in this study.


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Table 3. Summary of DNA microarray analysis of W3110Y cells exposed to 3 h, 7 h and 10 h nitrogen starvation

 
Among the 4000 genes tested, a large number of genes, including many functionally unknown (y) genes, showed significant alteration in their expression. The number of the altered genes is comparable to that of genes involved in the DNA damage response (Khil & Camerini-Otero, 2002). The number of induced genes in cells exposed to 3 h, 7 h and 10 h of nitrogen starvation were 318, 189 and 165, respectively, and the number of repressed genes were 503, 194 and 140, respectively (Table 3). Of the genes altered by 10 h nitrogen starvation, the top 30 up-regulated ones were nac, glnA, yeaG, cbl, amtB, 228#10 (GenBank no. P71569), argT, yhdW, yeaH, artJ, trpB, argG, asnB, yedL, potF, 228#6 (P96707), dppA, ynfM, hisJ, argC, intE, 228#9 (P74965), gdhA, yeaI, cstC, ntrB, asd, lysC, 228#7 (P26174) and ybeJ, and the top 30 down-regulated ones were dnaK, tnaA, ibpA, aspA, dctA, rbsB, yfiA, rbsD, ychH, ibpB, deoC, fxsA, slp, mopB, gatY, dnaJ, yiiS, gatD, yceP, yecI, gatC, gapA, hdeA, gatR, mopA, deoB, hyaA, ftsL, hdeB and dps. Alteration in expression of some of those genes was observed only at 7 h and 10 h or only at 10 h after the initiation of starvation. This may be due to different sensitivities of the genes to concentrations of intracellular nitrogen or may reflect secondary effects by changes in expression of other genes.

Starvation for 3 h seemed to repress a relatively large number of genes in all categories except for the {sigma}N regulon, whereas the starvation induced many genes, including {sigma}S regulon genes and ABC transporter genes (Table 3). Such a pronounced down-regulation may cause a sharp decline in cellular activities immediately after the initiation of nitrogen starvation. Most of the down-regulated genes, however, may not be related to the reduction in the number of c.f.u. because nitrogen starvation for a short period had little effect on the number of c.f.u. (Fig. 3). The fluctuation in expression of many genes caused by 3 h nitrogen starvation seems to be transient since their expression levels had been restored to the control level at 7 h after the initiation of starvation.

On the other hand, under the conditions of relatively long nitrogen starvation for 7 h or 10 h, which resulted in a decrease in the number of c.f.u. upon further incubation, several genes in the {sigma}H and {sigma}S regulons in addition to those encoding some ABC transporters were significantly down-regulated. Additionally, other genes repressed include ftsL, stpA and slp, which are related to cell division, DNA-binding and outer-membrane function, respectively. chpA and chpR were found as induced genes under all phases tested (data not shown), whose products were demonstrated to act as a growth inhibitor and its suppressor, respectively (Masuda et al., 1993). Some of these genes could play a crucial role in maintenance of cell viability (see below).

Nitrogen limitation increases the expression of the glnALG operon and other Ntr genes via a unique cascade including a two-component regulatory system (Blauwkamp & Ninfa, 2002; Reitzer & Schneider, 2001). In this study, we observed significant up-regulation of the glnALG operon and many Ntr genes at three different starvation periods. The genes for ABC transporters (argT-hisJMPQ, gltLK, glnHPQ, yhdWXYZ, oppABCD, dppBCD and potF) or proteins related to amino acid metabolism (glnA, trpB, argG, asnB, argC, gdhA, cstC, ntrB, asd and lysC) were also highly expressed. Of the upregulated genes, nac, amtB, argT, potF, hisJ, glnA and ntrB were previously characterized as Ntr genes (Zimmer et al., 2000). Additionally, the relA gene that responds to the reduction of amino acid concentration was significantly expressed in 3 h and 7 h starvation periods. This up-regulation may thus reflect the response of nitrogen limitation inside the cells. On the other hand, up-regulation of the genes encoding proteins for aromatic amino acid metabolism (mhpE, aroL, aroG, trpA, trpB, trpC, trpG, trpE, hisC and pheA) as shown in Table 3 seems to be due to induction in response to depletion of the corresponding amino acid in the medium.

Crucial functions of {sigma}H regulon genes in nitrogen starvation
Considering the irreversible decline in c.f.u. that occurs after nitrogen starvation, some of the genes that exhibited significantly low expression after 7 h and 10 h of nitrogen starvation are thought to be crucial for cell survival. Such genes include {sigma}S and {sigma}H regulon genes as shown in Table 4. The dnaK, ibpBA, mopB and clpB genes in the {sigma}H regulon encode chaperones Hsp70 and Hsp16, chaperonin GroES and proteinase, respectively, which are known to play key roles in protein folding and protein degradation. Other chaperones, including Hsp40 and Hsp90, and chaperonin GroEL were also weakly expressed in all three nitrogen-starvation periods.

To further confirm the effects of nitrogen starvation on expression levels of several {sigma}H regulon genes, significantly affected in DNA microarray, RT-PCR was performed using total RNAs from cultures of W3110Y, W3110N or YU635 (W3110N rpoS : : Tn10) that had been exposed to nitrogen starvation for 10 h (Table 5). The results of the RT-PCR in W3110Y are nearly consistent with those of the microarray in W3110Y and with those of the RT-PCR in YU635, whereas W3110N did not show any significant decrease in expression of those genes. It is worthwhile mentioning that this gene expression profile of W3110N is nearly similar, except for gadA and hdeB, to that of E. coli K-12 MG1655 obtained by DNA microarray in ammonium starvation (Soupene et al., 2003). Interestingly, in the situation that many {sigma}H regulon genes were severely down-regulated by nitrogen starvation, the expression of rpoH encoding {sigma}H only slightly decreased, suggesting that the translation rather than the transcription of rpoH is crucial for the expression of those {sigma}H regulon genes under such conditions.

Relationship between rpoS mutation and c.f.u. reduction in nitrogen starvation
As shown above, the {sigma}H regulon genes encoding chaperones and chaperonins appear to be expressed only weakly in rpoS mutant cells but normally in cells bearing a functional rpoS under nitrogen-starved conditions. Such a down-regulation of these genes in the rpoS mutant strain was hardly observed under normal growth conditions (Oshima & Mori, unpublished), and cells bearing the functional rpoS gene were capable of retaining the c.f.u. level under nitrogen-starved conditions (Fig. 2). These findings suggest that both the rpoS mutation and nitrogen starvation cause lowered expression levels of some {sigma}H regulon genes, and thus that {sigma}S directly or indirectly controls the expression of these {sigma}H regulon genes in nitrogen starvation.

Down-regulation of some of {sigma}S regulon genes was also confirmed by RT-PCR with RNAs from W3110Y and YU635 (Table 5). Among the {sigma}S regulon genes and other genes down-regulated, the reduction in expression of dps and stpA in all three starvation periods and the reduction of cbpA and himD in the 3 h starvation period may be characteristic because these gene products appear to be involved in the drastic change in nucleoid form in the stationary phase, which is believed to be one of cellular transformations for survival under nutrient-limited conditions (Ishihama, 1999). In particular, the down-regulation of dps (pexB) would affect cell survival because the dps-defective mutant was incapable of forming compact nucleoids in late stationary phase (Kim et al., 2004). Down-regulation of the following genes that may be involved in cellular defence under conditions such as nitrogen starvation was observed: hdeA and hdeB encoding acid-resistant proteins presumably with a chaperone-like function for periplasmic proteins (Gajiwala & Burley, 2000), rpoE encoding {sigma}E that is required for maintenance of extracytoplasmic proteins (Missiakas & Raina, 1997), and yiiT encoding a universal stress protein homologue. Furthermore, gadA and gadB encoding glutamate decarboxylase isozymes and gadE (yhiE) were strongly reduced. These three gene products together with HdeA and Slp seem to induce acid resistance by working co-operatively (Masuda & Church, 2003; Hommais et al., 2004). Since most of the above-mentioned genes are under the control of {sigma}S, rpoS mutants are assumed to fail in preparing for a nitrogen-starved situation, which may also be responsible for the cell death. On the other hand, hyaABCDF and glgS in the {sigma}S regulon, which were strongly affected, appear to have no crucial cellular roles in cell survival.

The stiA, stiB and stiC genes, which are essential for Salmonella typhimurium survival during simultaneous phosphate, carbon and nitrogen starvation, require {sigma}S for their positive or negative control, and stiB is induced by nitrogen starvation in the rpoS background (O'Neal et al., 1994). E. coli possesses several homologues of these starvation survival genes, but none of them was significantly altered in expression in W3110Y under our conditions.

In conclusion, for cell survival under nitrogen-starved conditions, {sigma}S may direct its regulon genes to appropriately adjust cellular activities, to deal with stresses caused by the starvation and to transform nucleoids, in which {sigma}H regulon genes may play important roles. Many E. coli K-12 strains, however, have acquired an amber mutation of the {sigma}S gene although the reason behind the acquisition has not been elucidated. Additionally, the discovery of a number of y genes whose expression level was altered by nitrogen elimination allows us to speculate that many functionally undefined genes are required for the survival or death process in nitrogen starvation. Further studies are needed to identify individual genes directly engaged in steps of decline in the number of c.f.u. under conditions of nitrogen starvation.


   ACKNOWLEDGEMENTS
 
We thank Osao Adachi, Kazunobu Matsushita, Hirohide Toyama, Hanae Izu and MD. Elias for their helpful discussion. This work was supported by a Grant-in-Aid for Basic Research from the Ministry of Education, Science and Culture of Japan (to M. Y.).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 2 January 2004; revised 30 April 2004; accepted 21 May 2004.



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